Clonal hematopoiesis of indeterminate potential (CHIP), identified in a majority of individuals over the age of 50, is a consequence of the acquisition of somatic mutations in hematopoietic stem cells (HSCs)1-4. These genetic variants lend a fitness advantage to the HSC allowing it to clonally expand and contribute to the peripheral blood1-3. Mutations associated with CHIP commonly occur in the DNA methylation regulator DNA (cytosine-5-)-methyltransferase 3 alpha (DNMT3A)1-4. Mouse models have demonstrated the conditional, loss- of-function of this enzyme (Dnmt3aKO) results in an increase in self-renewal of mutant HSCs when confronted with replication stress leading to their clonal expansion in the bone marrow as compared to their wild-type counterparts5. In humans, however, CHIP has been shown to be stable in people over the course of 10 years without an increase in the variant allele frequency detected in the peripheral blood3. We propose environmental pressures in addition to the genetic changes, are necessary to promote clonal expansion of CHIP-associated mutant HSCs. Other known stressors to the hematopoietic compartment include infectious and inflammatory stress and the resulting cytokine signaling9-16. The hypothesis of this proposal is that the pressure of interferon gamma-induced stress selects for the survival and expansion of Dnmt3aKO HSCs. To investigate the differential effects of IFN? upon mutant HSCs, murine Dnmt3aKO HSCs will be transplanted into bone marrow (BM) environments enriched or depleted of interferon gamma (IFN?), a cytokine that stimulates HSCs to proliferate as part of the immune response to infection (Aim 1). Chimeric BM transplants will be used to directly compare the survival and expansion of Dnmt3aKO and control HSCs within each individual BM microenvironment. The quantitation of mutant HSCs present in the BM months post-transplant will determine if IFN? promotes clonal hematopoiesis thereby revealing a fitness advantage of Dnmt3aKO HSCs. To ascertain a mechanism for the resistance to the functional attrition of elevated IFN?-signaling we will perform molecular assays delineating differences in IFN?-induced DNA damage and DNA repair in Dnmt3aKO and control HSCs (Aim 2). We will measure the presence of reactive oxygen species ROS in HSCs in response to IFN? via flow cytometry. To evaluate replicative stress, DNA replication will be directly visualized through DNA fiber fluorography to reveal replication fork progress rate during IFN?-induced cell division. To determine if DNA damage response innately varies between Dnmt3aKO and control HSCs, reporter plasmids will be nucleofected into BM cells to measure the efficiency of the homologous recombination and non-homologous end joining repair pathways. Together the proposed research aims will evaluate the role of inflammatory stress in clonal hematopoiesis leading to the identification of the first known environmental effect and a potential mechanism responsible for promoting the clonal advantage of Dnmt3aKO HSCs.